A. Chiral Cycloether Derivatives
A.1. Cyclodehydration Reaction
The use of cyclodehydration reactions is of importance in the chemical industry. This kind of reaction is, for example, widely used for the production of tetrahydrofuran from 1,4-butanediol and, more generally, for the production of various cycloethers.
One of the most cost-effective and atom-efficient processes for producing cycloethers is through the acid-catalyzed dehydration of alcohols. This reaction is usually carried out in liquid phase. The acid-catalyzed cyclodehydration yet presents the following drawbacks:                possibility of rearrangements of alcohols from primary to secondary alcohols, thereby giving mixtures of products,        requirement of separation and neutralization steps when the acid catalyst is in homogeneous phase,        production in batch or semi-batch reactors when using homogeneous catalysts and not in continuous flow reactors,        formation of thermodynamic and kinetic products due to long residence time in the reactor,        possible isomerization of chiral compounds due to acid conditions.        
Of all the abovementioned issues, the isomerization of chiral compounds into a mixture of stereoisomers is of particular concern. Indeed, the separation of different isomers constitutes a challenging task involving several steps, substantial product loss and extra costs that could be avoided by using a stereoselective synthesis pathway in the first place.
The issue of chirality is especially relevant when performing the cyclodehydration of 1,4- and 1,5-diols in the particular case when one of the two alcohols is a chiral tertiary alcohol and the second one is a primary alcohol. Given that tertiary alcohols are known to be more reactive to acid-catalyzed dehydration than primary or secondary alcohols, the dehydration of the chiral tertiary alcohol during the acid-catalyzed cyclodehydration process takes place readily through the formation of a carbocation, thereby disrupting the chiral configuration at the tertiary alcohol stereocenter. The cyclization that ensues therefore generates a mixture of isomers and most often of enantiomers.
An example of such issue can be found in the thesis of Allemann C., “Synthesis and Application of an Electronically Chiral Mimic of CpFe”, 2002, where the cyclodehydration of ambradiol to (−)ambrafuran under acidic conditions is said at page 33 to yield a racemic mixture of (−) and (+) ambrafuran (Scheme 1 below). Regarding the reasons of such isomerization, Allemann C. writes further that “the configuration at C-8 needs to be preserved during the intramolecular nucleophile substitution of the tertiary alcohol on the C-12 that bears the primary alcohol. Thus, the elimination of the 8-hydroxyl group must be prevented”.

Similarly, among other methods known to the art, one way to avoid the loss of the chiral configuration in the cyclodehydration of a diol when one of the two alcohols is a chiral tertiary alcohol and the other one is primary or secondary, consists in converting such primary or secondary alcohol into a better leaving group such as a mesylate, tosylate, methoxy methyl ether, phosphite ester or chlorosulfite group. Cyclization, usually under basic conditions, then takes place by attack from the tertiary alcohol on the substituted group, thereby preserving the original chiral configuration. Alternatively, ZnCl2 may be used to prevent elimination of the 8-hydroxyl group. However, such methods are not atom-efficient, result in chemical waste, use organic solvents of petrochemical source that contribute to increasing the environmental impact and energy cost of such transformations, use reagents that need to be separated from the final product and more generally contribute to high processing time and costs.
Therefore, there is a need for an improved stereoselective process for the cyclodehydration of Chiral Diols or Non-Soluble Diols.
A.2. Stereoselective Cyclodehydration Using Solid Catalysts
U.S. Pat. No. 3,029,255 and JP-A-86/33184 describe processes for the cyclization of ambradiol into (−)-ambrafuran using β-naphtalene sulphonic acid, alumina, bleaching earth, alumina or silica as catalysts. However, such methods generate undesirable secondary products, isomers around the 8th carbon stereocenter and result in low yields, generally below 70%.
U.S. Pat. No. 5,670,670 describes a process for the cyclization of ambradiol into (−)-ambrafuran using montmorillonite clay K catalysts with an acid charge below 80 or 100 mval at a temperature between 20° C. and 130° C.
Among smectite clays, montmorillonite and bentonites (together “MTM”) are defined by a layered structure formed by tetrahedral and/or octahedral sheets of approximate formula: Mx+(Si4-yAly)[(Al,Fe3+)2-z(Mg,Fe2+)z]O10(OH)2.nH2O where x=[0.2-0.6], x=y+z and y<<z.
MTMs are characterized as crystalline aluminosilicates with a large surface area while containing various other cations. They have been used as solid Bronsted and Lewis acid catalysts in their natural and ion-exchanged form to perform reactions such as ether or anhydride formation, esterification, Diels-Alder addition, rearrangements, oxidation-reduction and formylation. Also, MTMs have been successfully used for the dehydration of tertiary alcohols at moderate temperatures.
However, the method described in U.S. Pat. No. 5,670,670 requires the use of an organic solvent to solubilize the ambradiol, separation between the catalyst and the solvent containing the ambrafuran, further solvent drying and requires water content remaining below 2% for the cyclo-dehydration to take place, which conflicts with the release of water during the cyclo-dehydration process.
Patent application US2010/0248316 proposes an enantioselective cyclodehydration method consisting in exposing ambradiol to an activated zeolite at a temperature between 0° C. and 110° C. for a period of between 1 and 24 hours. This method allows the enantioselective cyclodehydration of the diol into (−)-ambrafuran. In this patent application, the cyclodehydration of ambradiol on activated zeolite is carried out in hexane or in toluene at room temperature or in dimethylsulfoxyde (DMSO) or ethyl acetate, optionally heating the solution. At the end of the reaction, solvent should be removed under reduced pressure.
Zeolites are aluminosilicates, which are well known as solid catalysts. The main features of these materials are their channel dimensions and stable structures. Their use in industrial processes generally results in a reduction in waste and pollution. The zeolite used in patent application US2010/0248316 is a Group II A metal zeolite, with calcium as metal.
In most situations, zeolites need to be activated before being used as catalysts, especially by removing from active sites water or other small molecules that may have been adsorbed. In US2010/0248316, one treatment consists in a reflux treatment at 90° C. for 24 hours in presence of ammonium nitrate. Another treatment is activation at 500° C. under vacuum or with a conventional microwave oven. After treatment, the activated zeolite should be kept in a closed container before use to avoid inactivation through contact with ambient air.
Several drawbacks remain when trying to apply US2010/0248316 to the stereoselective cyclodehydration of Chiral Diols or Non-Soluble Diols. One of them is the use of specific organic solvents to carry out the cyclodehydration reaction and to extract and purify the resulting product. Specifically, during the cyclodehydration step, only toluene and hexane are claimed to afford 100% conversion rate in 4 hours at room temperature while ethanol or ethyl acetate afford conversion rates of only 5.4% and 3.7% respectively. The cost of solvents, the manipulations needed as well as the evaporation steps are not optimal. Moreover, solvent use often results in atmospheric contamination during drying steps or disposal of spent material and may contaminate the final product, which is not environmentally friendly, requires costly recycling processes and may constitute health risks for consumers.
Another drawback of the method described in patent application US2010/0248316 is that the conversion requires between 1 and 4 hours to proceed fully.
Yet, another drawback of the method of patent application US2010/0248316 is the restrictive activation step of the zeolite performed at high temperature. The treatment described for zeolite activation further rules out the establishment of a fully continuous production process. Moreover, the manipulation of the activated zeolite from the drying site to the dehydration site automatically induces a partial inactivation of the zeolite through air contact, which in turn results in using a higher ratio of zeolite to ambradiol. In order to avoid zeolite inactivation and zeolite recharging, use would have to be performed in controlled airtight atmosphere, which inevitably contributes to generating extra costs compared to a minimal setup. Another drawback is the potential coking of the zeolite that may take place during reactivation at 500° C.
Also, zeolites have the capacity to catalyze a wide range of undesirable reactions such as dehydrogenation, esterification or Diels-Alder reactions. Specifically, time-dependent ambrafuran isomerization was also observed when using CBV320A during the dehydration of ambradiol. This implies the need for very strict control of reaction time and it seems impossible to avoid minimal isomer formation during the reaction.
A.3. Cyclodehydration of Targeted Diols Using HTW
The Applicant investigated the feasibility of performing (i) the stereoselective cyclodehydration of Chiral Diols into corresponding cycloether derivatives while retaining the initial chirality and/or (i) the cyclodehydration of Non-Soluble Diols into corresponding cycloether derivatives, in either case in a pressure-controlled and heated vessel containing HTW, optionally fed with supercritical fluids and gases such as CO2 and/or N2, surfactants, catalysts and other additives.
It was previously shown that the reversible cyclodehydration of 1,4-butanediol into tetrahydrofuran may be carried out in HTW (Richter T. and Vogel H., Chem. Eng. Technol., 2001, 24, 340-343; Hunter S., Ehrenberger C. and Savage P., J. Org. Chem., 2006, 71, 6229-6239). At the temperature and pressure conditions used in these references, the authors showed that the cyclodehydration of 1,4-butanediol into tetrahydrofuran is acid-catalyzed by H2O serving directly as a proton donor and/or by native H+ ions. Therefore, these conditions were never envisaged for the stereoselective synthesis of chiral cycloether derivatives from Chiral Diols as acid-catalyzed cyclodehydration is known to imply isomerization in such molecular configurations.
According to the Applicant's knowledge, no specific information can be found in prior art regarding the transformation using HTW of Chiral Diols into corresponding cycloether derivatives with retention of the initial chirality or regarding the cyclodehydration of Non-Soluble Diols.
The Applicant however carried tests using a pressure-controlled vessel containing HTW and surprisingly found that Chiral Diols could be irreversibly and stereoselectively cyclodehydrated into a corresponding cycloether derivative while retaining the original chirality and/or that Non-Soluble Diols could be irreversibly cyclodehydrated into a corresponding cycloether derivative, using HTW, preferably mixed with a supercritical fluid and/or gas such as CO2 and/or N2 and/or other additives, preferably while stirring such a mixture between 10 and 1500 rpm and/or while homogenizing the mixture with methods known to the art such as ultrasonication or homogenization and/or optionally in combination with other solvents, catalysts and/or surfactants. Moreover, it was found that temperatures and/or pressures below the ones described by Hunter et al. were efficient to carry out the cyclodehydration. In one embodiment, it was also found that in the conditions of the present invention, the cyclodehydration may be irreversible and therefore total conversion may be expected contrary to what was described in the prior art.
The stereoselectivity of the cyclodehydration reaction in HTW as well as the ability to perform such transformations regarless of the diol solubility in or miscibility with HTW were both surprising.
Indeed, in the case of Non-Soluble Diols, the heterogeneous catalytic dehydration of diols in HTW had not been found in prior art and could not be expected to take place. In the case of Chiral Diols, as mentioned above, it was shown that dehydration in HTW occurs through an acid catalysis involving protonation of the alcohol function and carbocation formation. Therefore, the cyclodehydration in HTW of Chiral Diols should have inevitably resulted in the disruption of the chiral configuration at the tertiary alcohol carbon site and thereby in the production of a mixture of isomers. Instead, in the present invention, using temperatures and pressures quite lower than in Hunter et al. and Richter et al surprisingly resulted in completely avoiding isomerization. Without willing to be bound by any theory, it is thought that at these temperatures and pressures, the cyclodehydration of Chiral Diols catalyzed by HTW may involve the linking of a hydrogen atom from a water molecule with the oxygen atom of the relevant tertiary alcohol in the substrate, such tertiary alcohol being positioned at the water-substrate interface in the case of non-HTW soluble compounds, thereby triggering a cyclodehydration mechanism. However, at these temperatures and pressures, the dehydration mechanism is not yet fully understood.
A.4. Cyclodehydration of Targeted Diols in Water Over a Solid Catalyst Such as for Example K-Type Montmorillonite Clay
Furthermore, the Applicant investigated the feasibility of performing in water at temperatures below HTW conditions (i) the stereoselective cyclodehydration of Chiral Diols into corresponding cycloether derivatives while retaining the initial chirality and/or (ii) the cyclodehydration of Non-Soluble Diols into corresponding cycloether derivatives, in both cases using a solid catalyst such as for example K-type montmorillonite, optionally ion-exchanged with a Class I or Class II metal or charged with Hydrogen.
The Applicant carried further experiments using montmorillonite clays and surprisingly found that Chiral Diols could be cyclo-dehydrated into their corresponding chiral cycloether derivative with retention of the initial chirality and/or that Non-Soluble diols could be cyclo-dehydrated into their corresponding cycloether derivative, in both cases in water medium over K-type montmorillonites where the total water weight content of the mixture is higher than 3%, preferably while stirring such a mixture and/or subjecting it to ultrasonication and/or optionally in combination with other solvents, catalysts and/or surfactants.
These findings are particularly surprising given that the absence of solubilization of a substrate in an appropriate solvent is known to increase mass transfer resistance in heterogeneous catalysis and should result in poor catalytic activity. Such findings are even more surprising given that high concentrations of water on the surface of montmorillonite are expected to reduce the activity of the dehydration catalyst. In that respect, U.S. Pat. No. 5,670,670 writes specifically that the water content of the reaction mixture should not be higher than 2% for the dehydration to take place. Furthermore, the addition of montmorillonite clays to water could be expected to increase Bronsted-type acidity, which would result in carbocation formation in place of the tertiary alcohol and in the subsequent isomerization of the final product.
A.5. Cyclodehydration of Targeted Diols in HTW Over a Solid Catalyst Such as for Example K-Type Montmorillonite Clay
The applicant surprisingly found that combining HTW with a solid catalyst such as for example K-type montmorillonites allowed to perform (i) the stereoselective cyclodehydration of Chiral Diols into corresponding cycloether derivatives while retaining the initial chirality and/or (ii) the cyclodehydration of Non-Soluble Diols into corresponding cycloether derivatives, in substantially shorter reaction times and/or with higher product selectivity than either using HTW alone or a solid catalyst such as for example K-type montmorillonites in water or in an organic solvent such as hexane. More specifically, using HTW in combination with a solid catalyst such as for example K-type montmorillonites allowed to improve the stereoselective cyclo-dehydration of ambradiol into (−)-ambrafuran from a yield of approximately 92.5% in 3.5 hours in pure HTW to a yield of 98% in 10 minutes in HTW mixed with montmorillonites.
Therefore, a solution found by the Applicant to operate the (i) the stereoselective cyclodehydration of Chiral Diols into corresponding cycloether derivatives with retention of the initial chirality and/or (ii) the cyclodehydration of Non-Soluble Diols into corresponding cycloether derivatives comprises performing such reaction using HTW, a solid catalyst such as for example K-type montmorillonites or a mixture thereof, preferably while keeping the total water weight content of the mixture above 3% in all operating conditions (see scheme 2 below).

Using HTW and/or a solid catalyst such as for example K-type montmorillonites in the present invention for performing the cyclodehydration of Chiral and/or Non-Soluble Diols, presents the following further advantages:                avoiding the use of conventional organic solvents and hence providing environmental benefits,        avoiding the use of chemical reactants and reducing the need to recharge catalysts at high temperatures between 300° C. and 550° C.,        when diols are not miscible with water, allowing their separation from water by simple decanting, centrifugation or filtration, thereby potentially avoiding drying, purification and solvent extraction steps,        making it possible to perform the transformation in a continuous process, thereby allowing for reduced processing time and fewer transformation steps.B. Manufacturing of Ambrafuran        
The Applicant found that one substrate of particular interest that can be synthesized by the process of the invention using a cyclodehydration step under HTW conditions and/or through contact with a solid catalyst such as for example K-type montmorillonite in water is dodecahydro-3a,6,6,9a-tetramethylnaphto[2,1-b]furan, also called ambrafuran, preferably (−)-ambrafuran.
Ambrafuran is marketed by Firmenich S.A. under the trademark Ambrox® and is an important fragrance chemical. This molecule, and especially (−)-ambrafuran, its Laevo isomer (3aR,5aR,9aS,9bS)-3a,6,6,9a-tetramethyl-dodecahydronaphto[2,1-b]furan, is responsible for the characteristic odor of ambergris—a naturally occurring amber note with a rich woody character—and is used as a fixative agent in perfumes. This fragrance is used in perfumery but also increasingly in hygiene products or detergents. The production of ambrafuran including its isomers is estimated to about 200 tonnes/year.
Ambergris is a metabolic substance produced in the digestive track of the sperm whale. When expelled from the whale, ambergris floats at the surface of the sea, being exposed to air and sunlight. These conditions led to changes in the substance and are essential to the development of the fragrance. Due to its origin, natural ambergris is extremely rare. Moreover, its commercial exploitation has been forbidden by the Washington Treaty.
B.1. Known Syntheses of (−)-Ambrafuran
Due to the value of ambergris, extensive research has been conducted to analyze its composition, revealing the importance of ambrafuran and especially its (−)-ambrafuran isomer. Several routes of chemical synthesis of (−)-ambrafuran were therefore explored.
Total syntheses were developed as well as hemi-syntheses, starting from naturally occurring sesqui- or di-terpens such as sclareol, a compound extracted from Salvia sclarea (clary sage).
The main drawback of these syntheses is the high number of steps needed to obtain ambrafuran, usually eight steps from sclareol as reported in US patent application US2010/0248316. The last steps of the chemical synthesis are reported below (scheme 3), showing the synthesis of the intermediate products sclareolide and ambradiol:

Chemical syntheses also imply the use of harsh conditions for certain steps (corrosive compounds, low temperature, toxic and dangerous reactants) that would be preferably avoided. Also, the use of organic solvents in chemical syntheses presents several drawbacks such as: drying costs, potential toxicity for consumers of solvent residues in the final product, lack of solvent selectivity resulting in the presence of unwanted compounds in the final product. Another concern with chemical syntheses of ambrafuran is that they generally result into racemic mixtures instead of isolated enantiomers. Moreover, consumers are more and more asking for “green products” obtained by environmentally friendly processes compatible with sustainable growth. One of the objectives of sustainable growth consists in avoiding the use of chemical reagents and the rejection of residual salts in the environment, which is incompatible with conventional syntheses of ambrafuran.
An alternative to chemical hemi-synthesis of ambrafuran from sclareol consists in replacing the first six or seven steps of the traditional transformation process leading respectively to sclareolide and to ambradiol intermediates with a biological conversion step.
The biological conversion of sclareol to sclareolide and ambradiol was first reported in U.S. Pat. No. 4,798,799 for the synthesis of ambradiol and in U.S. Pat. No. 4,970,163 for the synthesis of sclareolide and ambradiol.
The biological conversion of sclareol to sclareolide described in U.S. Pat. No. 4,970,163 uses the microorganisms Cryptococcus albidus saito, skinner var. albidus, ATCC 20918 or Cryptococcus albidus, ATCC 20921. Such biotransformation is carried out under aerobic conditions, in an aqueous nutrient medium containing sclareol. Further isolation and purification of sclareolide may be achieved by conventional techniques such as filtration or centrifugation, by solvent extraction, distillation or crystallization. A method for purifying sclareolide is reported in U.S. Pat. No. 5,945,546. Once extracted and purified from the fermentation broth, sclareolide may then be converted into ambradiol by hydrogenation as reported in the eight-step chemical synthesis.
The biological conversion of sclareol to ambradiol is described in U.S. Pat. No. 4,798,799 using the microorganism Hyphozyma roseoniger (CBS214.83 and ATCC 20624) and in U.S. Pat. No. 4,970,163 using the microorganisms Bensingtonia Ciliata ATCC 20919 or Cryptococcus Laurentii ATCC 20920. Such biotransformations are carried out under aerobic conditions at 20° C., in an aqueous nutrient medium containing sclareol.
In U.S. Pat. No. 4,798,799, ambradiol is then dehydrated to give ambrafuran directly from the aqueous nutrient medium or after recovery. More specifically, recovery and purification of ambradiol may be achieved by conventional techniques such as filtration or centrifugation, by solvent extraction, distillation or crystallization. Especially, ambradiol may be extracted with ethyl acetate and crystallized from hexane/chloroform.
The conversion of ambradiol to (−)-ambrafuran may then be performed by conventional cyclization methods, such as by reacting ambradiol with toluene-p-sulfonylchloride in pyridine at 0° C., followed by solvent extraction. Alternatively, dehydration is achieved in the presence of an acid catalyst, most often in the form of a strong mineral acid, heteropolyacids, sulfonic acids or DMSO. One of the drawbacks of such methods is that in acidic conditions, ambrafuran is likely to isomerize readily to the more thermodynamically stable, but olfactively much weaker iso-ambrafuran. Also, such methods do not solve the problems associated with chemical synthesis as they also contribute to undesired environmental and economic burdens through the rejection of waste salts, the need to regenerate the catalyst or the use of organic petrochemical solvents.
As already mentioned above, patent application US2010/0248316 proposes an alternative cyclization method consisting in exposing ambradiol to an activated zeolite (scheme 4). This method allows the dehydration of the diol into (−)-ambrafuran, ambradiol being obtained by the biological conversion disclosed in U.S. Pat. No. 4,798,799. Therefore, starting from racemic natural sclareol, this enantioselective method achieves the production of a single enantiomer of ambrafuran in two steps.

In patent application US2010/0248316, the dehydration of ambradiol on an activated zeolite is carried out in hexane or in toluene at room temperature or in dimethylsulfoxyde or ethyl acetate, optionally heating the solution. At the end of the reaction, solvent should be removed under reduced pressure.
Contrary to other dehydrations that need controlled temperatures, patent application US2010/0248316 discloses a total conversion at room temperature, generally in less than two hours.
Despite advantages of the method disclosed in US2010/0248316 such as the suppression of toxic reactants, several drawbacks remain that were detailed in part A above, the main ones being:                the use of specific organic solvents to extract and purify ambradiol from the fermentation broth, to carry out the dehydration reaction and to extract and purify (−)-ambrafuran;        the restrictive activation step of the zeolite.B.2. Use of the Process of the Invention for Manufacturing of Ambrafuran        
Therefore, there is a need for an improved process for the production of products of interest selected from the group comprising sclareolide, ambradiol and/or ambrafuran from sclareol presenting the following characteristics:                avoiding the use of synthetic solvents or organic solvents of petrochemical origin at any step of the transformation process, in particular during purification or transformation steps, and/or        avoiding the use of chemical reactants that result in the production of salts and other potentially toxic or polluting spent material, and/or        using a continuous process to reduce the time of transformation and number of steps involved, and/or        simplifying the conditions of purification and/or conversion of sclareol, sclareolide and ambradiol leading to the production of (−)-ambrafuran.        
The Applicant found that the stereoselective cyclodehydration process of the present invention may be incorporated into an integrated manufacturing process of ambrafuran without the use of petrochemical organic solvents or chemical reactants. Especially, the Applicant showed that ambradiol may be converted into (−)-ambrafuran of high purity through the process of the invention.
Moreover, in this invention, the Applicant identified a complete process for manufacturing ambrafuran from sclareol comprising an initial biological conversion of sclareol and at least one further step performed according to the cyclo-dehydration process of the present invention.
Given that the biological conversion of sclareol may provide ambradiol and/or sclareolide, the invention also relates to the purification of sclareolide and/or ambradiol from a bioconversion broth.
Therefore, the Applicant showed that sclareolide and/or ambradiol may be efficiently purified from a bioconversion broth using supercritical CO2 with or without other cosolvents such as ethanol or methanol.
These elements were not foreseeable and required experimental work to be established.
B.3. Supercritical Conditions Applied to Ambradiol and/or Sclareolide Purification
The Applicant carried out research for applying supercritical fluids to the, purification of sclareolide and/or ambradiol from a bioconversion broth and, surprisingly, showed that sclareolide and ambradiol may be efficiently separated from a bioconversion broth using supercritical CO2 with or without cosolvent.
Generally, the ability to extract and purify substances using supercritical CO2 is difficult to predict without experimentation. The efficiency of supercritical extraction and purification depends altogether on the characteristics of the targeted substance such as polarity and molecular mass, on the temperature and pressure parameters of the supercritical fluid, but also on the characteristics of the medium containing the substances to be extracted, such as composition, polarity, granularity, etc. In weak solubility or affinity conditions, the addition of a cosolvent such as methanol or ethanol is often necessary but may result in lower selectivity. Therefore, the ability to extract substances in high purity from a medium using supercritical fluids is to be explored on a case-by-case basis and conditions of pressure and temperature have to be optimized for each substance or mix thereof.
Whether or not sclareolide and mostly ambradiol were soluble in supercritical carbon dioxide with or without cosolvent and whether the selectivity of their extraction from a bioconversion broth was acceptable in regard of the overall production process was therefore not foreseeable; to the contrary, in view of the polarity brought by the two hydroxyl functions in ambradiol and of the presence of oxygen atoms in sclareolide, the skilled artisan was induced to estimate that solubility of these compounds in supercritical carbon dioxide was mostly questionable. For example, initial experiments in dry form revealed very poor solubility of both compounds in pure supercritical CO2 whereas solubility of ambradiol in pure supercritical CO2 proved to be enhanced when extraction was performed from an aqueous medium such as a bioconversion broth.
B.4. Evaluation of the Feasibility of Supercritical Purification of Sclareolide and Ambradiol from Bioconversion Broth
Furthermore, the ability to extract sclareolide or ambradiol in high purity from a bioconversion broth required practical experimentation in order to test the relative affinity of the targeted substances for supercritical CO2 with and without cosolvent versus their aqueous medium or the potential pollution of the supercritical extracts by additives contained in the bioconversion broth such as growth factors, nutrients, substrates or by-products produced by the organisms or biocatalysts.
Tests were therefore carried out by the Applicant and it was found that the extraction of sclareolide or ambradiol by supercritical CO2 with and without cosolvent from the aqueous medium used for the biological conversion was possible in high purity, even in the presence of several additives such as growth factors, nutrients and substrates, all of which could have retained ambradiol or sclareolide within the broth or polluted the extract.
The Applicant further found that several of the compounds used in yeast nutrient mediums or as co-factors in the case of biocatalysis are not soluble in supercritical CO2 with and without cosolvent and/or that their affinity for the supercritical solvent mixture did not allow their efficient extraction from the bioconversion broth. Some of the bioconversion broth additives identified by the Applicant in regard of the existing art include without being limited to: enzymes, carbon sources such as glucose, galactose, L-sorbose, maltose, sucrose, cellobiose, trehalose, L-arabinose, L-rhamnose, ethanol, glycerol, L-erythrithol, D-mannitol, lactose, melibiose, raffinose, melezitose, starch, D-xylose, D-sorbitol, a-methyl-D-glucoside, lactic acid, citric acid, succinic acid; organic sources of nitrogen such as peptone, meat extract, yeast extract, corn steep liquor, casein, urea, amino acids but preferably inorganic sources of nitrogen such as: nitrates, nitrites, inorganic ammonium salts; inorganic salts such as phosphates of magnesium, potassium, calcium or sodium; several vitamins but preferably vitamin B1, B2, B3, B5, B6, B7, B9, B12; minerals such as Fe, Mo, Cu, Mn and B as well as most acids, preferably inorganic acids, used to adjust broth pH. In general, the Applicant found that such additives showed poor solubility in supercritical CO2 with or without cosolvent at the operating conditions used for the extraction of sclareolide and/or ambradiol.
Therefore, the Applicant established that ambradiol and/or sclareolide may be purified from a bioconversion broth with up to 99% purity and 100% yield, using one or two steps of supercritical CO2 extraction.
When sclareolide is produced, transformation into ambradiol may then be performed by methods known to the art, such as by using metal hydrides such as LiAlH4 or RedAl®.
B.5. Evaluation of the Feasibility of the Synthesis of Ambrafuran in HTW
The Applicant also investigated, as shown in FIG. 4, the feasibility of the conversion of ambradiol into ambrafuran using HTW.
Ambradiol is non-miscible with and non-soluble in water or HTW up to around 250° C., at which point ambradiol is degraded by HTW. At the same time, ambradiol also bears a chiral tertiary alcohol which is likely to lose its chirality during acid-catalyzed dehydration. Therefore, ambradiol conforms with both definitions of a Chiral Diol and a Non-Soluble Diol.
The use of HTW conditions was never envisaged for the synthesis of ambrafuran from ambradiol. Indeed, catalytic mechanisms are difficult to predict and the sensitivity of reactants and products might have prevented reaching the temperatures needed to operate cyclo-dehydration reactions in HTW.
The use of HTW altogether as a reaction medium, a solvent and a catalyst in dehydration transformations is documented in JOC Article “Kinetics and Mechanism of Tetrahydrofuran Synthesis via 1,4-Butanediol Dehydration in High-Temperature Water”, where Hunter, Ehrenberger and Savage describe the dehydration of the diol 1,4-butanediol (BDO) to the furan tetrahydrofuran (THF) using sub-critical water (HTW) at temperatures between 200° C. and 350° C. However, according to Applicant's knowledge, no specific information can be found in prior art regarding the stereoselective transformation of ambradiol into (−)ambrafuran using HTW.
Due to the configuration of the ambradiol molecule, one skilled in the art, in view of the above article, may conclude that using HTW on ambradiol could lead to numerous possible transformations. Also, at ambient temperature, BDO and THF are liquids miscible with water, whereas ambradiol consists of a white solid that melts around 132° C. while remaining immiscible with water in HTW. No indication was found in the prior art as regards the ability of ambradiol to form a unique phase with HTW, the impact of HTW on such a molecule at a given temperature as well as the very stability of such a molecule at the temperatures potentially leading to the envisaged transformations.
The Applicant however carried tests using a pressure-controlled vessel containing HTW and noticed that ambradiol could be dehydrated into (−)ambrafuran, the primary isomeric conformation of interest, in high yield and, more surprisingly, with a purity up to about 92.5% using HTW, preferably mixed with a supercritical fluid and/or gas such as CO2 and/or N2 and/or other additives, preferably while stirring such mixture between 10 and 1500 rpm and/or while homogenizing the mixture with methods known to the art such as ultrasonication or homogenization.
B.6. Evaluation of the Synthesis of Ambrafuran Using a Solid Catalyst Such as for Example Montmorillonite Clays in Water
The Applicant also investigated, as shown in FIG. 3, the feasibility of the conversion of ambradiol into ambrafuran using a solid catalyst such as for example K-type montmorillonite clays in water.
Given that ambradiol is a Non-Soluble and Chiral Diol, water had never been used as a solvent for performing such a transformation. Moreover, U.S. Pat. No. 5,670,670 requires specifically that water content should remain below 2% when performing the cyclodehydration of ambradiol using montmorillonite clays.
Surprisingly, the Applicant found that ambradiol could be efficiently cyclo-dehydrated into (−)ambrafuran with a purity above 90% using a solid catalyst such as for example K-type montmorillonite clays, preferably when the reaction mixture had a water content above 3% and, more generally, above 80%, between room temperature and 90° C.
Furthermore, when combining a solid catalyst such as for example montmorillonite clays and HTW conditions, the Applicant found that (−)ambrafuran could be produced from ambradiol at close to 98% yield and in above 95% purity in less than 15 minutes by using preferably montmorillonite clays when the reaction mixture had a water content above 3% and, more generally, above 80% and while subjecting further the reaction mixture to HTW conditions.
Therefore, the Applicant identified a transformation process that can not only achieve the purification of sclareolide and/or ambradiol from a bioconversion broth but also the chemical cyclodehydration of ambradiol into (−)-ambrafuran.
The Applicant found that not only supercritical conditions were useful for the purification of intermediate compounds, i.e. sclareolide and/or ambradiol, but also that HTW conditions and/or a solid catalyst such as for example K-type montmorillonites with high water content could be used for the chemical conversion of ambradiol into (−)-ambrafuran. These elements are summarized in FIG. 1.